Abstract This proposal describes a 5-year research plan focusing on how signal transduction through the T cell receptor (TCR ) contributes to the homeostasis and expansion of regulatory T cells (Treg)s. Tregs are a subset of T cells with suppressive function and are crucial for preventing immunopathology by inhibiting conventional T cell (Tconv) activation. A failure to develop Tregs or to maintain Tregs in the periphery results in fatal autoimmune disease. In contrast, an increase in Tregs protects against a variety of T cell-mediated disorders including graft-versus-host disease (GVHD). Thus, understanding the mechanisms by which Tregs proliferate and are maintained is of critical importance. However, the precise cellular and molecular requirements for Treg homeostasis and expansion are incompletely understood. We have recently repored that Tconvs and dendritic cells (DC)s play a critical role in controlling Treg homeostasis and expansion. Our preliminary data suggest that (1) Tregs require DCs but not TCR stimulation for their proliferation, (2) TCR stimulation of Tconvs by DCs for IL-2 production is required for Treg proliferation, and (3) blockade of TCR stimulation in conjunction with exogenous IL-2 inhibits Tconv activation but maintains Treg proliferation. These data support our hypothesis that the homeostatic maintenance and proliferation of Tregs depend on Tconvs and DCs, and occur through both TCR/MHC II-dependent and - independent routes. We propose that TCR stimulation allows the preferential expansion of Tregs with relevant antigen specificities, while the MHC II/TCR-independent mode of Treg proliferation ensures survival of Tregs with TCRs of multiple specificities to continuously maintain a diverse repertoire of Tregs. In this proposal, we aim to extend our preliminary in vitro findings to in vivo studies. Our research plan starts by examining the precise contribution of TCR signaling by Tregs to their homeostasis in vivo. Our second aim will test the role of TCR signaling by Tconvs for IL-2 production in supporting Treg homeostasis in vivo. Finally, we will translate our hypothesis to clinically relevant questions by exploiting the differential requirement of TCR signaling in Treg and Tconv proliferation and determine whether a combination of IL-2 and a TCR signaling inhibitor such as CSA could be used to more effectively treat Tconv-mediated diseases such as GVHD. The proposed studies will enhance our understanding of Treg homeostasis and potentially yield novel molecules, strategies, and pathways to modulate Tregs in multiple disease settings. Our expertise in signal transduction and Treg biology together with our expert consultants (Drs. Andy Caton, Gary Koretzky, Robert Eisenberg, and Andras Schaffer), and the quality of the research facilities at the University of Pennsylvania Medical Center comprise an ideal environment in which to conduct this proposed research plan.